Gram-negative pathogens can rapidly alter the composition of proteins in their envelope to adapt to a wide range of stresses, including challenges by the immune system and antibiotic treatment. A lack of knowledge of the molecular mechanisms by which these bacteria regulate the expression of envelope proteins limits understanding of interactions between pathogen and host, and prevents targeting crucial systems for development of new drugs. Our overall strategy is to identify inhibitors of molecular interactions required for the sE cell envelope sensing pathway, the major pathway used by Gram-negative bacteria to regulate outer membrane protein composition in response to challenges. This pathway is essential for viability in several important Gram-negative pathogens and required for virulence in others. The long-term goal of our research is to elucidate the role and mechanism of action of the sE pathway in bacterial physiology and pathogenesis. The objective of this proposal, which is the next required step in the attainment of our goal, is to develop an assay for high-throughput screening (HTS) to identify small molecule inhibitors of two key steps in the sE pathway: sE-dependent transcription, and regulation of porin mRNA levels by the protein Hfq in conjunction with sE-dependent sRNAs. Studies of genetic mutations in components of this pathway have led to a basic understanding of how the pathway functions. However, this approach is limited because it is difficult to distinguish direct functions of the pathway from the pleiotropic effects caused by the mutations. Small molecule inhibitors will enable experiments to test the function and molecular mechanism of this important pathway in greater detail. Guided by strong preliminary data, our objective will be attained by pursuing two specific aims: 1) optimize an assay for high-throughput screening to identify molecules that specifically inhibit two key components of the CE pathway;and 2) adapt and develop assays for secondary screening. Under the first aim, a primary assay that has been developed and tested in a pilot screen will be optimized for use in a high-throughput format. Under the second aim, existing assays will be adapted for use in secondary screens to eliminate false positive hits and to identify the molecules targeted by each inhibitor. The assays will be submitted to Molecular Libraries Production Centers Network (MLPCN) for implementation. Information about small molecule-target interactions obtained from these studies will be made available via PubChem. This proposal is innovative in its approach to simultaneously target a transcription factor, sE, and sRNA regulators that are required for a concerted physiological response to environmental challenges. All components of this pathway are highly conserved, so it is anticipated that inhibitors identified here can be used to study the role of this pathway in growth and virulence of many pathogens. These inhibitors will also provide lead compounds for future antibiotic development. PUBLIC HEALTH RELEVANCE: The proposed study will develop assays for high-throughput screening to identify inhibitors of a conserved bacterial pathway required to survive environmental and immune response challenges. Successful completion of these experiments and subsequent screening will provide a crucial set of reagents for testing how bacteria interact with their surroundings during growth and virulence. These reagents may also be lead compounds for development of new antibiotics.